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es el 23-09-2016

Tangarife-Castao V, Correa-Royero J, Zapata-Londoo B, et al.


Anti-Candida albicans activity, cytotoxicity and

interaction with antifungal drugs of essential oils
and extracts from aromatic and medicinal plants
Actividad contra Candida albicans, citotoxicidad e interaccin
con antifngicos de aceites esenciales y extractos de plantas medicinales
y aromticas

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Objective: To determine anti-Candida albicans activity, cytotoxicity and drug interaction of essential oils and extracts from plants collected in
Materials and methods: The antifungal activity was evaluated following the AFST-EUCAST protocol. With most active samples, the inhibition of the
formation of germ tubes and budding, the in vitro pharmacodynamics, using time-kill assays, and the interaction with itraconazole and amphotericin
B following the chequerboard technique were evaluated. The cytotoxicity assay for all samples was done using MTT.
Results: Strong activity in 17.57% of the samples was found. The lowest MIC values were obtained with Piper bredemeyeri Jacq and Lippia origanoi-
des Kunth (B) oils and Morinda royoc L extract. The three samples inhibited the formation of germ tubes and budding. P. bredemeyeri Jacq oil and M.
royoc L extract samples showed fungicidal activity at 2xMIC. A synergistic effect was obtained with the combination of P. bredemeyeri Jacq oil and
itraconazole, but not for the combination with amphotericin B. Active samples against C. albicans were not cytotoxic on Vero cells ATCC CCL-81,
excluding P. bredemeyeri Jacq oil.
Conclusions: The results of this study suggest that Colombian medicinal and aromatic plants represent an untapped source of compounds with
anti-C. albicans activity that could be a resource in the development of new therapeutic natural products.
Key words: Essential oils, extracts, Candida albicans, antifungal activity, cytotoxicity, synergism

Objetivo. Determinar la actividad anti-Candida albicans, la citotoxicidad y la interaccin con antifngicos de aceites y extractos de plantas recolec-
tadas en Colombia.
Materiales y mtodos. La actividad antifngica fue evaluada siguiendo el protocolo Antifungal Susceptibility Testing Subcommittee of the European
Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST). Con las muestras ms activas se evalu la inhibicin de la formacin de tubo
germinal y la gemacin, la farmacodinamia mediante curvas de tiempo muerte y la interaccin con itraconazol y anfotericina B. Se determin la
citotoxicidad mediante la tcnica MTT.
Resultados. Se encontr actividad en 17,57 % de las muestras. La mayor actividad se obtuvo con los aceites de Piper bredemeyeri Jacq y Lippia origanoides
Kunth (B) y el extracto de Morinda royoc L. Las tres muestras inhibieron la formacin de tubo germinal y la gemacin. El aceite de P. bredemeyeri Jacq y el
extracto de M. royoc L mostraron actividad fungicida con dos veces la concentracin inhibitoria mnima. Se encontr un efecto sinrgico por la combinacin
del aceite de P. bredemeyeri Jacq e itraconazol, pero no con anfotericina B. Las muestras activas no fueron citotxicas, excepto el aceite de P. bredemeyeri Jacq.
Conclusin. Los resultados de este estudio sugieren que las plantas de Colombia son una fuente no explorada de compuestos con actividad anti-C.
albicans, tiles para el desarrollo de nuevos productos teraputicos.
Palabras clave: aceites esenciales, extractos, Candida albicans, actividad antifngica, citotoxicidad, sinergismo.

Introduction pounds in medicinal plants. Screening by using

in vitro evaluation is a useful tool for the disco-
Many researchers, particularly the ones from very of new potential antifungal agents from na-
countries with a rich biodiversity, have contri- tural products such as essential oils and extracts
buted to the detection of new antifungal com- derived from plants (1). Colombia is the second ri-

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Anti-Candida albicans effect, cytotoxicity and interaction with antifungal drugs of essential oils and extracts
from aromatic and medicinal plants
chest country in the world in biodiversity, and its Materials and methods

vascular plants (2). Although Colombia possesses Plant material

a rich tradition in the use of medicinal plants, the Stems and leaves of 74 plants were collected
antifungal activity of medicinal plants derivates in different regions of Colombia, from 2005 to
has not been deeply studied. 2008, as part of a survey conducted by CENI-
VAM, a research center devoted to the study of
Candidiasis is a common infection of the skin, aromatic plants and essential oils in Colombia.
nails, oral cavity, esophagus, and vagina, caused
by yeast of the Candida genus. Systemic yeast samples was performed by Jos Luis Fernandez
infections are a common consequence of immu- at the Herbario Nacional de Colombia, Instituto
de Ciencias Naturales, Facultad de Ciencias, Uni-
nosuppression, long-term indwelling catheters,
versidad Nacional de Colombia at Bogot, whe-
and endocrinopathies. Candida albicans is the
re voucher specimens were deposited.
most common pathogen causing that fungal
opportunistic infection. Additionally, it has the
The selected plants belong to the following gene-
ability to adhere to host surfaces or to prosthesis
ra: Lippia (Verbenaceae) (26); Salvia (Lamiaceae)
(7); Lepechinia (Lamiaceae) (3); Piper (Piperaceae)
ther facilitate adhesion, infection and resistance
(3); Baccharis (Asteraceae) (2); Hedyosmum (Chlo-
to the antifungals (3). ranthaceae) (3); Illicium (Schisandraceae) (2);
Cymbopogon (Poaceae) (3); Minthostachiys mollis
In Colombia, different studies have shown the (Lamiaceae) (2); Thymus vulgaris (Lamiaceae) (2);
importance of C. albicans as the principal agent and Tquoctkpwu"qhekpcnku"(Lamiaceae) (2). Addi-
causing bloodstream fungal infection (44.7%) in tionally, the following plants were also selected:
tertiary care level hospitals (4), and as cause of Cananga odorata (Annonaceae), Tagetes lucida
invasive infection (43.6% and 54.5%) (5,6). (Asteraceae), Eucalyptus citriodora (Myrtaceae),
Turnera aff. diffusa Willd. ex Schult (Turneraceae),
The number of clinical infections worldwide by Nectandra tomentella (Lauraceae), Sigesbeckia
C. albicans has risen considerably in recent years, agrestis (Asteraceae), Lantana fucata Lindl (Ver-
and the incidence of resistance to traditional an- benaceae), Hyptis perbullata (Lamiaceae), Ori-
tifungal therapies is also increasing (7). In addition, ganum vulgare (Lamiaceae)." kpikdgt" qhekpcng"
(Zingiberaceae), Cascarilla saravena (Euphorbia-
ceae), Achyrocline alata (Kunth) DC (Asteraceae),
antifungals, have encouraged the search for new Pimenta racemosa (Myrtaceae), Elettaria carda-
momum L (Zingiberaceae), Aloysia triphylla (Ver-
alternatives among natural products (8).
benaceae), Curcuma monteria (Zingiberaceae),
Chenopodium ambrosioides L (Amaranthaceae),
The aim of this study was to evaluate anti-C. al-
Bursera graveolens Bur-
bicans activity and cytotoxicity of essential oils
seraceae), and Morinda royoc L (Rubiaceae).
and plant extracts obtained from aromatic and
medicinal plants of different Colombian regions.
Extracts and essential oils extraction
Furthermore, the combined effects of itracona-
zole and amphotericin B with the most actives Essential oils (59) and extracts (15) were evalua-
samples, and their pharmacodynamics were ted. The essential oils were extracted from dried
evaluated in vitro by the chequerboard method stems and leaves (300 g) by microwave-assisted
and the time-kill curves, respectively. hydrodistillation as described (9). The extracts

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Tangarife-Castao V, Correa-Royero J, Zapata-Londoo B, et al.

were obtained from 40 g of dried leaves from -

each plant, macerated with 200 ml ethanol and bia) and cells without treatment, respectively,
left in suspension for 7 days at room temperatu- were included.
trated using a Buchi rotavapor. Stock solutions of For germ tube assays, after two hours of incu-
both oils and extracts, were prepared in DMSO bation at 35 C, the absence or presence of germ
tubes was determined using a light microscopy
(Nikon, Eclipse E200, Japan). Each sample was
Antifungal susceptibility testing tested in duplicate using two different assays. To
evaluate the effect on budding, after three hours
The Minimum Inhibitory Concentrations (MIC) of of incubation at 35 C, the budding cells/ml were
all samples were determined by the Antifungal counted using a hemacytometer.
Susceptibility Testing Subcommittee of the Eu-
ropean Committee on Antibiotic Susceptibility The statistical analysis of the effect on budding,
Testing (AFST-EUCAST) protocol (10). Candida al- was performed by R version 2.9.1 (The R Foun-
bicans ATCC 10231, C. albicans ATCC 90028 and dation for Statistical Computing, ISBN 3-900051-
a clinical blood isolate randomly selected from 07-0) using ANOVA after transforming the
a group of clinical isolates of the Candida spe- response variable with square root. The Tukey
cies collected in our laboratory were used at multiple comparison test was used to compare
strains with and without treatment. A 0.05 signi-
inoculum size of 1-5 x 105 CFU/ml. The oils and

Time-kill assay
sidered active when they exhibited MIC values

The in vitro pharmacodynamics of P. bredeme-

(Sigma-Aldrich, Co., MO, USA) was used as a po-
yeri Jacq oil and M. royoc L extract against C.
albicans ATCC 90028 were performed by the
the strains C. krusei ATCC 6258 and C. parapsi-
method described by Klepser, et al.(12). An ini-
losis ATCC 22019. A negative control (inoculum
tial inoculum ranging from 1-5 x 105 CFU/ml
without treatment) was also included. MIC va-
was seeded on each sample, at concentrations
lues were expressed as a geometric mean (GM-
of 0.25, 0.5, 1 and 2 times the MIC. The sam-
MIC) of tests performed in duplicate in three di-
ples were incubated at 35 C with agitation. At
fferent assays against each Candida strain and
0, 4, 8, 12, and 24 hours, volumes of 10 l were
the clinical isolate. then spread onto potato dextrose agar (Oxoid,
Basingstoke, Hampshire, UK) and incubated at
Effect on germ tube formation and budding 37 C for 24 hours to determine the number of
CFU/ml. The limit of detection was 100 CFU/ml.
The effect on germ tube formation and budding Time-kill curves with itraconazole and amphote-
was evaluated according to Ishida, et al. .(11). Di- ricin B (Sigma-Adrich, Co, MO, USA) were used
P. as fungistatic and fungicide controls, respecti-
bredemeyeri Jacq, L. origanoides Kunth (B) oils vely. Experiments were carried out in duplicate
and M. royoc L extract were tested with the three in two separate experiments. Time-kill curves
yeasts. A suspension of 1-5 x 105 CFU/ml in RPMI were constructed by plotting of mean stan-
dard deviation (SD) of colony count (log10 CFU/
added to the same volume of each sample con- ml) as a function of time (hours) with the Pris-
centration. Positive and negative controls with ma (GraphPad Software, Inc., USA, 2007) sta-

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Anti-Candida albicans effect, cytotoxicity and interaction with antifungal drugs of essential oils and extracts
from aromatic and medicinal plants
bredemeyeri Jacq (GM-MIC of 157.5, 176.8 and
from 222.7 g/ml for C. albicans ATCC 10231, C. al-
the starting inoculum (0.5 2.5 x 10 CFU/ml). bicans ATCC 90028 and the clinical isolate, res-
pectively) and L. origanoides Kunth (B) (GM-MIC
Interaction of essential oils and drugs C. albicans ATCC
10231, C. albicans ATCC 90028, and the clinical
Assays were performed using the chequerboard isolate, respectively). Also M. royoc L extract was
method (13). Candida. albicans ATCC 10231 was
CFU/ evaluated yeasts (Table 1).The MIC values of the
positive control itraconazole with C. parapsilosis
ATCC 22019 and C. krusei ATCC 6258 (0.169 and
P. bredemeyeri 0.999, respectively), were in the acceptable ran-
ge according to the standard protocol (0.3125-
inhibitory index (FICI) was calculated, and the in-
nergistic effect; >0.5 but <4 no interaction; and Piper bredemeyeri Jacq and L. origanoides Kunth
. (B) oils inhibited the formation of germ tubes
in the two strains and in the clinical isolate at a
Citotoxicity assay M. royoc L
extract inhibited the formation of germ tube in
Cercopithecus aethiops African green monkey both: C. albicans ATCC 90028 strain and in the cli-
kidney cells (Vero cell line ATCC CCL-81) were nical isolate, at the same concentrations. The in-
used. The cells were grown in Eagles minimum hibition in C. albicans
essential medium (MEM) supplemented. The g/ml concentration. Germ tubes were observed
cytotoxicity of the essential oils and their com- in the negative control, but not in the positive
ponents was examined in vitro using an MTT control. In addition, the treatment of the strains
(dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium and the clinical isolate with P. bredemeyeri Jacq
bromide) (Sigma, New Jersey, USA) assay as des- and L. origanoides Kunth (B) oils, and M. royoc
cribed by Betancur-Galvis, et al..(14). The minimal extract, inhibited the process of budding, at 250
dilution of the essential oils that induced 50% g/ml. The decrease of budding in treated blas-
growth inhibition of the cells was expressed as toconidia, compared with the negative control
inhibitory concentration 50% (IC50). The IC50 va-
lues for each compound were obtained by linear control inhibited the budding, and in the negative
regression analysis of the dose-response curves control there was a normal rate of budding.
generated from the absorbance data with the R
(Development Core Team, Vienna, Austria, 2008) The killing activity of P. bredemeyeri Jacq oil and M.
statistical package. IC50 values were expressed as royoc L extract against C. albicans ATCC 90028 as
the mean standard deviation (M SD) of two well as itraconazole and amphotericin B is repre-
independent experiments done in quadruplicate. sented in Figure 1. At 2 times the MIC, P. bredeme-
yeri Jacq oil, after 4 hours of incubation, showed
Results fungicidal activity similar to amphotericin B (Figu-
re 1a and Figure 1c, respectively). It was found that
The MICs of 13 active samples (GM-MIC range at 0.5 times and 1 time the MIC concentration the-
157.5-500 g/ml) tested against C. albicans and re was a reduction in growth until approximately
the respective IC50 values, are presented in Table 8 hours, when colony counts continued to grow
1. The most active samples were the oils from P. near the initial starting inoculum. M. royoc L ex-

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Tangarife-Castao V, Correa-Royero J, Zapata-Londoo B, et al.

tract showed fungicidal activity at 2 times the MIC In this study, oils and extracts from medicinal
after 8 hours of incubation (Figure 1b). At lower and aromatic plants were tested against two C.
concentrations (0.25, 0.5 and 1 times the MIC), we albicans strains and a clinical isolate. Currently,
(<3 - there is no agreement on the acceptable level
log10) compared with the starting inoculum. of activity for natural products when they are
compared to standard drugs. Aligiannis, et al.(16),
A synergistic effect was obtained for the combi-
nation of itraconazole and P. bredemeyeri Jacq in plant derivates (antifungal activity based on
(FICI range 0.09-0.13), but no interaction was MIC results) as follows: strong inhibitors, MIC up
detected for the combination of P. bredemeyeri to 0.5 mg/ml; moderate inhibitors, MIC between
Jacq and amphotericin B (FICI=1.06). 0.6 and 1.5 mg/ml; weak inhibitors, MIC above
1.6 mg/ml. According to these criteria, we found
According to the American National Cancer Ins-
strong anti-C. albicans activity in 17.57% of the
titute (USA) criteria samples with anti-C. albicans
evaluated samples (Table 1).
activity were not cytotoxic on Vero cells excluding
P. bredemeyeri Jacq oil (IC50=15.2 3) (Table 1).
Previous studies have shown the antifungal ac-
tivity in extracts and oils of plants belonging to
Piper, Morinda, and Lippia genus (17-19). Lpez, et
al.(19), determined a MIC value of 100.000 g/
Plants, marine organisms, and microbes usua-
ml against C. albicans of the methanolic extract
lly produce biologically active compounds as a
from the plant Piper lanceafolum, using an agar
defense against predators and competition with
neighbors. Thus, it seems logical that most of diffusion method. In contrast, in this study, the
the drugs derived from natural sources have an- microdilution standard method AFST-EUCAST
ticancer or anti-infective properties. Important was used, and lower MICs with P. bredemeyeri
antifungal agents such as polyenes, echinocan- Jacq were found (GM-MIC of 157.5, 176.8, and
dins/pneumocandins, aureobasidins and sorda- 222.7 g/ml, with the three evaluated yeasts)
rins had their origin in natural products (15). (Table 1).

a b
10 10

8 8
Log 10 CFU/mL

Log 10 CFU/mL

6 6

4 4

2 2

0 0
0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28
Time (h) Time (h)
c d
10 10

8 8
Log 10 CFU/mL

Log 10 CFU/mL

6 6

4 4

2 2

0 0
0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28
Time (h) Time (h)

Figure 1. Time-kill plots against C. albicans ATCC 90028 of a) P. bredemeyeri Jacq oil, b)
M. royoc L y P. bredemeyeri
control; 0.25x MIC; 0.5x MIC; 4x MIC

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Anti-Candida albicans effect, cytotoxicity and interaction with antifungal drugs of essential oils and extracts
from aromatic and medicinal plants
Table 1. Geometric means of minimal inhib itory concentration (GM MIC, g/mll) and inhibitory concentration 50% (IC50) of essential oils and
extracts actives against Candida albicans

GM - MIC (g/ml) IC50 (M SD) R2

Voucher C. albicans C. albicans C. albicans

Plant Sample Vero ATCC CCL-81
specimen ATCC 10231 ATCC 90028 (Clinical isolate)
Lippia alba 007 484650 Oil * 353.6 420.4 32.8 3.6 0.89

Lippia origanoides 512271 Oil 396.9 500 500 52.3 11.5 0.8

Lippia origanoides 512075 Oil 250 250 315 74.6 16.9 0.75

Lippia origanoides 512075 Oil 500 500 500 60.4 11.2 0.83

Piper bredemeyeri Jacq. 516939 Oil 157.5 176.8 222.7 15.2 3 0.81
Turnera aff. diffusa Willd. ex
516293 Oil * 353.6 500 52.2 5.2 0.93
Lippia origanoides Kunth (A) 517741 Oil 500 500 500 31.4 5.6 0.75

Lippia origanoides Kunth (B) 517741 Oil 157.5 157.5 198.4 31.4 5.6 0.75

Lippia origanoides Kunth (C) 517741 Oil 500 396.9 396.9 34.4 5.9 0.76

Morinda royoc L. 512222 Extract 250 250 250 NA

Piper hispidum Sw. 519969 Oil 250 280.6 250 51.7 9.3 0.8

Lippia origanoides 512087 Oil 500 500 500 104.4 5.9 0.98

Cymbopogon citratus 531013 Oil * 500 500 NA

* MIC>500 g/ml; R2

This is one of the few studies focused on evalua- The concentration at which P. bredemeyeri Jacq
ting the anti-C. albicans activity of L. origanoides and L. origanoides Kunth (B) oils were able to
oils using a standard method for determination inhibit the germ tube formation of C. albicans
of MIC by broth dilution of fermentative yeast.
Oliveira, et al .(20), also found important activi- concentration ranges published for this activity
ty against C. albicans in an oil of L. origanoides with Melaleuca alternifolia oil (tea tree oil) (0.25
using an agar diffusion method; however, we and 12.5% (v/v, approximately 2.25 and 112.5
cannot compare our results with theirs, because g/ml, respectively) (24). Those results make our
they used a technique that determined inhibi-
tion zone (mm) but not MIC (g/ml). oil is used as commercial product for treatments
of fungal infections as oropharingeal and vagi-
Candida albicans is a dimorphic fungus, able to nal infections by C. albicans (24-26).

hyphal transition begins with the formation of Candida albicans has isotropic growth by budding
a germ tube and it is the initial stage of hyphal and several antifungal drugs and plant extracts can
formation (21). The oils from P. bredemeyeri Jacq inhibit budding of yeast cells (21, 27). In this study the
and L. origanoides Kunth (B) and the M. royoc L treatment of the strains and the clinical isolate with
extract inhibited the germ tube formation. It is P. bredemeyeri Jacq and L. origanoides Kunth (B)
possible that those oils and extracts are acting oils, and M. royoc extract, decreased blastoconidia
on an important process in the morpho-trans- budding at 250 g/ml compared with the negative
formation of C. albicans, as wall or membrane control (p<0.05). Previous studies have suggested
integrity or reorganization of the cytoskeleton (22, that the deleterious effect of the essential oil on the
. This characteristic could make a new antifun- cell wall of the fungus could be the main reason for
gal more selective towards the infecting germ the decrease in the rate of yeast budding, because
than towards the host cells. the cell wall is necessary for cell division (27).

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The time-kill studies are useful for the evaluation and C. parapsilosis has been demonstrated. Un-
of the pharmacodynamics characteristics of new like P. bredemeyeri Jacq oil only showed activity
antimicrobial agents (28). The oil of P. bredemeyeri against C. krusei (unpublished data).
Jacq and the extract of M. royoc L showed a fun-
gicidal activity on C. albicans at 2 x MIC after 4 Potential synergy of essential oils with antibiotics
and 8 hours, respectively. It is clinically more im- has been previously considered with the aims of
- increasing the rate of fungal killing, shortening
gistatic, particularly in HIV patients, because the the duration of therapy, avoiding the emergence
prophylactic use of fungistatic drugs has been of drug resistance, expanding the spectrum of
associated with an increased frequency of innate activity, and decreasing drug-related toxicity by
or acquired drug resistance in clinical isolates (29). allowing lower doses of antifungal agents to be
administered (33). In this study, a synergistic effect
Chemical analyses of most active essential oils was obtained when itraconazole and the oil from
against C. albicans have been carried out in our P. bredemeyeri Jacq were combined (FICI range,
laboratory. The main compounds of P. bredeme- 0.09-0.13). No interaction was detected with the
yeri Jacq oil were terpenes alpha-pinene (20.3%) combination of the oil of P. bredemeyeri Jacq and
and beta-pinene (32.3%) (unpublished data). amphotericin B (FICI=1.06).
Other studies have demonstrated that those ter-
The criteria of cytotoxic activity for the crude ex-
penes act on cellular integrity, inhibition of the
tracts as established by the American National
respiration and ion transport processes, and in-
Cancer Institute (USA), is an IC50 of less than 30
crease membrane permeability in C. albicans (30,
g/ml (34). According to these criteria, we consi-
. Hence, it is possible that the antifungal acti-
der that oils with anti-C. albicans activity and M.
vity of P. bredemeyeri Jacq oil could be explained
royoc L extract were not cytotoxic on Vero cells
by a higher concentration of those monoterpe-
(IC50 P. bredeme-
ne hydrocarbons. On the other hand, chemical
yeri Jacq (IC50=15.2 3 g/ml) (Table 1). Gonz-
analyses of oil from L. origanoides Kunth (B) have
lez, et al..(35), orally administered an extract of M.
also been carried out in our laboratory. The main
components were the oxygenated monoterpenes
Both our results of in vitro citotoxicity, as well as
thymol (43.8%) and carvacrol (17.3%). The antimi-
the toxicity in vivo demonstrated by Gonzlez, et
crobial activities of those terpenes have also been
al..(35), suggest that M. royoc L extract may be a
demonstrated, and their mechanism of action has candidate for developing an antifungal of natu-
been associated with membrane permeability (20). ral origin against C. albicans.

The discovery of a novel natural product for

time that antifungal activity of M. royoc L extract therapeutic use is a slow process. For example,
is described. Morinda genus contains substan- Taxol, an anticancer agent developed from the
tial amounts of anthraquinones, especially in the plant Taxus brevifolia, was discovered after a
roots. The antifungal activity of those quinones random screening of 35,000 plant samples that
has been demonstrated (32), so it is possible that took more than 25 years (36, 37).
anti-C. albicans activity of M. royoc L extract may
be produced by the presence of such molecules. In conclusion, the results of this study suggest that
Colombian medicinal and aromatic plants repre-
In our laboratory, the antifungal activity of L. sent an untapped source of compounds with anti-
origanoides Kunth (B) and M. royoc L extract C. albicans activity that could be a resource in the
against Aspergillus fumigatus,"C0"cxwu."E0"mtwugk" development of therapeutically natural products.

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Anti-Candida albicans effect, cytotoxicity and interaction with antifungal drugs of essential oils and extracts
from aromatic and medicinal plants
Acknowledgements 17. Duarte MC, Figueira GM, Sartoratto A, Rehder VL, Delarmelina C.
Anti-Candida activity of Brazilian medicinal plants. J Ethnopharma-
col. 2005;97:305-11.
Grant RC-245-2011 (Fondo Nacional de Finan- 18. Holetz FB, Pessini GL, Sanches NR, Cortez DA, Nakamura CV, Filho
BP. Screening of some plants used in the Brazilian folk medicine
ciamiento para la Ciencia, la Tecnologa y la In- for the treatment of infectious diseases. Mem Inst Oswaldo Cruz.
novacin, Francisco Jos de Caldas). 2002;97:1027-31.
19. Lpez A, Hudson JB, Towers GH. Antiviral and antimicrobial activi-
ties of Colombian medicinal plants. J Ethnopharmacol. 2001;77:189-
References 20. Oliveira DR, Leitao GG, Bizzo HR, Lopes D, Alviano DS, Alviano CS
et al. Chemical and antimicrobial analyses of essential oil of Lippia
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